5, (1)*Target (protein/gene name): succinate dehydrogenase *NCBI Gene # or RefSeq#:
Symbol: PF10_0334
Gene ID: 810491 *Protein ID (NP or XP #) or Wolbachia#: PlasmoDB / PF10_0334GeneDB / PF10_0334 *Organism (including strain): Plasmodium falciparum Etiologic Risk Group (see link below): Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents *Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Plasmodium falciparum is a parasite that is one of the most common causes of malaria. In 2002, it was estimated that 2.2 billion people were exposed to malaria, resulting in approximately 515 million clinical attacks of this disease. The majority (~70%) of incidence of this disease in 2002 occurred in Africa, with a substantial incidence rate (~25%) in Southeast Asia [1]. A global map depicting the endemicity of malaria in 2007 showed high endemicity in Africa, low endemicity with pockets of mid to high endemicity in Central/Southeast Asia, and uniformly low endemicity in the Americas [2].
Because malaria is generally concentrated in lower income countries, the ideal treatment option for malaria would be cheap, single-dose and have minimal side effects. However, the current treatment options fall short of meeting this criteria. For decades, choroquine (CQ) was the main treatment option for malaria; however, as years passed, CQ-resistant malaria developed. Today, CQ is no longer a viable treatment option for most instances of malaria. Other drugs used in malaria treatment have serious side effects. As such, there is a pronounced need for novel anti-malarial drugs [3].
Essentiality of this protein:
This protein is essential because according to the journal article regarding purification of the protein, the SDH enzyme would be a good drug target for antimalarial drugs. Complex of proteins?: no Druggable Target: Yes-druggability is 1
--Show screenshot of BRENDA enzyme mechanism schematic Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
spectrophotometric -- link to Sigma (or other company) page for assay or assay reagents (substrates): no information available on Sigma -- link (or citation) to paper that contains assay information http://www.sciencedirect.com/science/article/pii/S0166685199001802
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 97%
---- Max % Identities: 62%
---- % Positives: 74%
---- Chain used for homology: Chain A, crystal structure of mitochondrial respiratory complex li from porcine heart at 2.4 Angstroms
Current Inhibitors:
Inhibitors for the protein have not been tested in P. falciparum, but in E. coli, there are 11 known inhibitors for succinate dehydrogenase. Expression Information (has it been expressed in bacterial cells) http://www.ncbi.nlm.nih.gov/pubmed/15847182
SDH has been expressed in E.coli. Purification Method: http://www.sciencedirect.com/science/article/pii/S0166685199001802
Fast protein liquid chromatographic system Image of protein (PyMol with features delineated and shown separately): *Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MIRNNKKYIRFMQSSFCRFSNIKTKAYDIIDHHYDAVIVGAGGAGLRSALELSKNKYKVA CISKLFPTRSHTVAAQGGINAALGNMTEDDWRWHAYDTIKGSDWLGDQNAIHYMCREAPD SVLELEEFGLPFSRTKDGKIYQRAFGGQSLKYGKGGQAYRCAAAADRTGHAMLHTLYGQS LSYNCIFFVEYFVLDLLMLNSNECIGVICINIADGKIHRFFTPHTVIATGGYGRAYLSCT SAHACTGDGNAIVARSKLPLQDLEFVQFHPTGIYPAGCLITEGCRGEGGILRNKEGEAFM MRYAPKAKDLASRDVVSRAMTIEINEQRGCGPNADHIYLDLTHLPYETLKERLPGIMETA KIFAGVDVTKQYIPVLPTVHYNMGGIPTNYKTQVLTQNVNFNKQTNKSNEDIIVKGLYAA GEAASASVHGANRLGANSLLDIVVFGKRAALTIMEIDKPNIPKINANTNIGEESIQRLDH IRFNKGSIQTSQLRKKMQICMQKHAAVFRIGPLLQEGYKQILEICSIFKDIEITDKTLTW NTDLLETLELENLLTLASQTILAAVERKESRGAHARDDFPERDDKNYLKHSLTWMTDRNI ENTKYFTTYRDVITKPLDNEMEYVPPVKRV *length of your protein in Amino Acids 631 amino acids Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite
70694.8 kDa Molar Extinction coefficient of your protein at 280 nm wavelength:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 70095 Abs 0.1% (=1 g/l) 0.992, assuming all pairs of Cys residues form cystines
Ext. coefficient 69220 Abs 0.1% (=1 g/l) 0.979, assuming all Cys residues are reduced
*CDS Gene Sequence (paste as text only): ATGATTAGGAATAATAAAAAATACATAAGATTTATGCAATCAAGTTTTTGTAGATTCTCAAATATTAAAA CGAAAGCATATGATATAATTGATCATCATTATGATGCAGTAATTGTAGGAGCTGGAGGTGCTGGATTAAG ATCTGCATTGGAATTATCAAAAAATAAATACAAGGTAGCATGTATCAGTAAATTGTTTCCAACACGATCA CATACTGTAGCTGCTCAAGGTGGAATAAATGCAGCTTTAGGTAATATGACTGAAGATGATTGGAGATGGC ATGCTTATGATACTATCAAAGGTTCAGATTGGCTTGGGGATCAAAACGCTATTCATTATATGTGTAGAGA AGCTCCTGATTCTGTATTAGAATTAGAAGAATTTGGACTCCCGTTTTCAAGAACAAAAGATGGGAAAATA TATCAAAGAGCTTTTGGAGGACAAAGTTTAAAATATGGTAAAGGAGGACAAGCTTATAGATGTGCTGCCG CTGCTGATAGAACAGGACATGCCATGTTACATACATTATATGGACAATCCTTATCTTACAATTGTATATT TTTTGTAGAATATTTTGTTTTAGATTTACTTATGTTAAATTCTAATGAATGTATTGGGGTAATCTGTATT AATATAGCAGATGGAAAAATACATAGATTTTTTACACCACATACTGTTATAGCTACTGGGGGATATGGTC GAGCTTATTTGTCTTGTACATCTGCTCATGCATGTACAGGAGATGGTAATGCCATTGTAGCTAGAAGTAA ATTACCATTACAAGATTTAGAATTTGTACAATTTCATCCAACAGGTATATACCCAGCTGGATGCTTAATT ACTGAAGGATGTAGAGGAGAAGGTGGTATTTTAAGAAATAAAGAAGGAGAAGCCTTTATGATGAGATATG CACCTAAAGCAAAAGATTTAGCTAGTCGTGATGTTGTTAGTAGAGCTATGACCATAGAAATTAATGAACA AAGAGGATGTGGACCAAACGCAGATCATATATATTTAGATTTAACACATTTACCATATGAAACATTAAAA GAAAGATTACCAGGCATAATGGAAACTGCAAAAATTTTTGCGGGAGTTGATGTAACTAAACAATATATTC CTGTCTTACCAACAGTTCATTATAATATGGGTGGAATTCCAACTAATTATAAAACACAAGTGTTAACACA AAACGTAAATTTTAATAAACAAACTAATAAATCAAATGAAGATATTATTGTAAAAGGCCTTTATGCTGCA GGAGAAGCTGCATCAGCATCTGTTCATGGAGCAAATCGGTTAGGAGCTAATTCACTTTTAGATATTGTCG TTTTTGGTAAAAGAGCTGCACTAACTATTATGGAGATAGATAAACCAAATATTCCTAAAATAAATGCAAA TACTAATATAGGTGAAGAATCTATACAAAGATTAGATCATATAAGATTTAATAAAGGTAGTATACAAACA TCGCAATTAAGAAAAAAAATGCAAATATGCATGCAAAAACATGCTGCTGTTTTTAGAATTGGACCGTTAT TACAAGAAGGTTATAAACAAATTCTTGAAATATGTTCCATTTTTAAAGACATAGAAATTACTGATAAAAC GTTAACATGGAATACAGATTTATTAGAAACATTAGAACTTGAAAATTTACTAACACTCGCATCACAAACT ATCTTAGCAGCTGTTGAAAGAAAAGAATCAAGAGGTGCTCATGCTCGTGACGATTTCCCTGAAAGAGATG ATAAAAACTATTTAAAACATTCCTTAACATGGATGACTGACAGAAATATTGAGAATACAAAATATTTTAC GACTTACAGGGATGTTATAACAAAACCATTAGATAATGAAATGGAATATGTTCCACCTGTTAAACGTGTT TATTAA *GC% Content for gene:
32% *CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now) Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
[1] Snow, R.W.; Guerra, C.A.; Noor, A.M.; Myint, H.Y.; Hay, S.I., The global distribution of clinical episodes of plasmodium falciparum malaria. Nature2005, 434, (7030), 214-217.
*NCBI Gene # or RefSeq#:
Symbol: PF10_0334
Gene ID: 810491
*Protein ID (NP or XP #) or Wolbachia#:
PlasmoDB / PF10_0334 GeneDB / PF10_0334
*Organism (including strain): Plasmodium falciparum
Etiologic Risk Group (see link below): Appendix B-II-C. Risk Group 2 (RG2) - Parasitic Agents
*Background/Disease Information (sort of like the Intro to your Mini Research Write up):
Plasmodium falciparum is a parasite that is one of the most common causes of malaria. In 2002, it was estimated that 2.2 billion people were exposed to malaria, resulting in approximately 515 million clinical attacks of this disease. The majority (~70%) of incidence of this disease in 2002 occurred in Africa, with a substantial incidence rate (~25%) in Southeast Asia [1]. A global map depicting the endemicity of malaria in 2007 showed high endemicity in Africa, low endemicity with pockets of mid to high endemicity in Central/Southeast Asia, and uniformly low endemicity in the Americas [2].
Because malaria is generally concentrated in lower income countries, the ideal treatment option for malaria would be cheap, single-dose and have minimal side effects. However, the current treatment options fall short of meeting this criteria. For decades, choroquine (CQ) was the main treatment option for malaria; however, as years passed, CQ-resistant malaria developed. Today, CQ is no longer a viable treatment option for most instances of malaria. Other drugs used in malaria treatment have serious side effects. As such, there is a pronounced need for novel anti-malarial drugs [3].
Essentiality of this protein:
This protein is essential because according to the journal article regarding purification of the protein, the SDH enzyme would be a good drug target for antimalarial drugs.
Complex of proteins?: no
Druggable Target: Yes-druggability is 1
*EC#:
1.3.99.1
Link to BRENDA EC# page:
http://www.brenda-enzymes.org/php/result_flat.php4?ecno=1.3.99.1
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assay information (spectrophotometric, coupled assay ?, reagents):
spectrophotometric
-- link to Sigma (or other company) page for assay or assay reagents (substrates): no information available on Sigma
-- link (or citation) to paper that contains assay information
http://www.sciencedirect.com/science/article/pii/S0166685199001802
http://www.sciencedirect.com/science/article/pii/S0005272813000893 (E. coli)
-- List cost and quantity of substrate reagents and supplier:
http://www.sigmaaldrich.com/catalog/product/sigma/p9625?lang=en®ion=US
Structure Available (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model:
-- For Homology Model option:
---- Show pairwise alignment of your BLASTP search in NCBI against the PDB
---- Query Coverage: 97%
---- Max % Identities: 62%
---- % Positives: 74%
---- Chain used for homology: Chain A, crystal structure of mitochondrial respiratory complex li from porcine heart at 2.4 Angstroms
Current Inhibitors:
Inhibitors for the protein have not been tested in P. falciparum, but in E. coli, there are 11 known inhibitors for succinate dehydrogenase.
Expression Information (has it been expressed in bacterial cells)
http://www.ncbi.nlm.nih.gov/pubmed/15847182
SDH has been expressed in E.coli.
Purification Method:
http://www.sciencedirect.com/science/article/pii/S0166685199001802
Fast protein liquid chromatographic system
Image of protein (PyMol with features delineated and shown separately):
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MIRNNKKYIRFMQSSFCRFSNIKTKAYDIIDHHYDAVIVGAGGAGLRSALELSKNKYKVA CISKLFPTRSHTVAAQGGINAALGNMTEDDWRWHAYDTIKGSDWLGDQNAIHYMCREAPD SVLELEEFGLPFSRTKDGKIYQRAFGGQSLKYGKGGQAYRCAAAADRTGHAMLHTLYGQS LSYNCIFFVEYFVLDLLMLNSNECIGVICINIADGKIHRFFTPHTVIATGGYGRAYLSCT SAHACTGDGNAIVARSKLPLQDLEFVQFHPTGIYPAGCLITEGCRGEGGILRNKEGEAFM MRYAPKAKDLASRDVVSRAMTIEINEQRGCGPNADHIYLDLTHLPYETLKERLPGIMETA KIFAGVDVTKQYIPVLPTVHYNMGGIPTNYKTQVLTQNVNFNKQTNKSNEDIIVKGLYAA GEAASASVHGANRLGANSLLDIVVFGKRAALTIMEIDKPNIPKINANTNIGEESIQRLDH IRFNKGSIQTSQLRKKMQICMQKHAAVFRIGPLLQEGYKQILEICSIFKDIEITDKTLTW NTDLLETLELENLLTLASQTILAAVERKESRGAHARDDFPERDDKNYLKHSLTWMTDRNI ENTKYFTTYRDVITKPLDNEMEYVPPVKRV
*length of your protein in Amino Acids 631 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParamwebsite
70694.8 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 70095 Abs 0.1% (=1 g/l) 0.992, assuming all pairs of Cys residues form cystines
Ext. coefficient 69220 Abs 0.1% (=1 g/l) 0.979, assuming all Cys residues are reduced
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (paste as text only):
ATGATTAGGAATAATAAAAAATACATAAGATTTATGCAATCAAGTTTTTGTAGATTCTCAAATATTAAAA CGAAAGCATATGATATAATTGATCATCATTATGATGCAGTAATTGTAGGAGCTGGAGGTGCTGGATTAAG ATCTGCATTGGAATTATCAAAAAATAAATACAAGGTAGCATGTATCAGTAAATTGTTTCCAACACGATCA CATACTGTAGCTGCTCAAGGTGGAATAAATGCAGCTTTAGGTAATATGACTGAAGATGATTGGAGATGGC ATGCTTATGATACTATCAAAGGTTCAGATTGGCTTGGGGATCAAAACGCTATTCATTATATGTGTAGAGA AGCTCCTGATTCTGTATTAGAATTAGAAGAATTTGGACTCCCGTTTTCAAGAACAAAAGATGGGAAAATA TATCAAAGAGCTTTTGGAGGACAAAGTTTAAAATATGGTAAAGGAGGACAAGCTTATAGATGTGCTGCCG CTGCTGATAGAACAGGACATGCCATGTTACATACATTATATGGACAATCCTTATCTTACAATTGTATATT TTTTGTAGAATATTTTGTTTTAGATTTACTTATGTTAAATTCTAATGAATGTATTGGGGTAATCTGTATT AATATAGCAGATGGAAAAATACATAGATTTTTTACACCACATACTGTTATAGCTACTGGGGGATATGGTC GAGCTTATTTGTCTTGTACATCTGCTCATGCATGTACAGGAGATGGTAATGCCATTGTAGCTAGAAGTAA ATTACCATTACAAGATTTAGAATTTGTACAATTTCATCCAACAGGTATATACCCAGCTGGATGCTTAATT ACTGAAGGATGTAGAGGAGAAGGTGGTATTTTAAGAAATAAAGAAGGAGAAGCCTTTATGATGAGATATG CACCTAAAGCAAAAGATTTAGCTAGTCGTGATGTTGTTAGTAGAGCTATGACCATAGAAATTAATGAACA AAGAGGATGTGGACCAAACGCAGATCATATATATTTAGATTTAACACATTTACCATATGAAACATTAAAA GAAAGATTACCAGGCATAATGGAAACTGCAAAAATTTTTGCGGGAGTTGATGTAACTAAACAATATATTC CTGTCTTACCAACAGTTCATTATAATATGGGTGGAATTCCAACTAATTATAAAACACAAGTGTTAACACA AAACGTAAATTTTAATAAACAAACTAATAAATCAAATGAAGATATTATTGTAAAAGGCCTTTATGCTGCA GGAGAAGCTGCATCAGCATCTGTTCATGGAGCAAATCGGTTAGGAGCTAATTCACTTTTAGATATTGTCG TTTTTGGTAAAAGAGCTGCACTAACTATTATGGAGATAGATAAACCAAATATTCCTAAAATAAATGCAAA TACTAATATAGGTGAAGAATCTATACAAAGATTAGATCATATAAGATTTAATAAAGGTAGTATACAAACA TCGCAATTAAGAAAAAAAATGCAAATATGCATGCAAAAACATGCTGCTGTTTTTAGAATTGGACCGTTAT TACAAGAAGGTTATAAACAAATTCTTGAAATATGTTCCATTTTTAAAGACATAGAAATTACTGATAAAAC GTTAACATGGAATACAGATTTATTAGAAACATTAGAACTTGAAAATTTACTAACACTCGCATCACAAACT ATCTTAGCAGCTGTTGAAAGAAAAGAATCAAGAGGTGCTCATGCTCGTGACGATTTCCCTGAAAGAGATG ATAAAAACTATTTAAAACATTCCTTAACATGGATGACTGACAGAAATATTGAGAATACAAAATATTTTAC GACTTACAGGGATGTTATAACAAAACCATTAGATAATGAAATGGAATATGTTCCACCTGTTAAACGTGTT TATTAA
*GC% Content for gene:
32%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Do Not Need this info for Spring (but still copy these lines to your Target page for now)
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
Resources:
See ProtocolTargetDiscoveryVDS.docx for moreEtiologic Risk Group Categories (for pathogens): http://www.utexas.edu/research/rsc/ibc/agent_class.html#_Toc7238334
Databases of genes/organisms:
http://www.niaid.nih.gov/Pages/default.aspx
http://eupathdb.org/eupathdb/
https://patricbrc.vbi.vt.edu/portal/portal/patric/Home
http://www.nmpdr.org/FIG/wiki/view.cgi/Main/EssentialGenes
http://tubic.tju.edu.cn/deg/
http://csgid.org/csgid/cake/pages/community_request_gateway
http://tdrtargets.org/
http://gsc.jcvi.org/status.shtml
Scientific Nomenclature page from Center for Disease Control (gene, protein names and abbreviations)
http://wwwnc.cdc.gov/eid/pages/scientific-nomenclature.htm
Gene Information:
NCBI GENE Page: http://www.ncbi.nlm.nih.gov/gene
BLAST Page: http://blast.ncbi.nlm.nih.gov/
Protein Information:
NCBI Protein Page: http://www.ncbi.nlm.nih.gov/protein
Protein Expression Website
Protein Expression Paper: SGC_ProteinProductionPurificationNatMethods2008.pdf
Primer Overlap PCR Articles
HooverLubkowski_PCRoverlapcloninggnf042.pdf
StemmerPCRoverlapGene1995.pdf
Is my target good for Virtual Screening programs?
Reynolds_THermodynamicsLigandBinding_MedChemLett2011.pdf
References
[1] Snow, R.W.; Guerra, C.A.; Noor, A.M.; Myint, H.Y.; Hay, S.I., The global distribution of clinical episodes of plasmodium falciparum malaria. Nature 2005, 434, (7030), 214-217.
[2] Hay, S.I.; Guerra, C.A.; Gething, P.W.; Patil, A.P.; Tatem, A.J.; Noor, A.M.; Kabaria, A.M.; Manh, B.H.; Elyazar, I.R.F.; Brooker, S.; Smith, D.L.; Moyeed, R.A.; Snow, R.W., A world malaria map: Plasmodium falciparum endemicity in 2007. PLoS Med 2009, 6, (3).
[3] Bell, D.J.; Molyneux, M.E., Treatment of childhood plasmodium falciparum malaria: Current challenges. Expert Review of Anti-Infective Therapy 2007, 5, (1), 141.